Diagnosis of tauopathies

ABSTRACT

The present invention provides a method for the diagnosis of tauopathies in an individual and/or for the differential diagnosis of a tauopathy versus a non-tauopathy based on the detection of the ratio of phospho-tau (181)/total tau in said individual. The present invention further provides a phospho-peptide for standardization in a method of the invention.

[0001] This is a divisional of co-pending application Ser. No.09/769,180, filed 24 Jan. 2001, which claims priority to EP 00870008.0,filed 24 Jan. 2000; U.S. provisional application Serial No. 60/178,391,filed 27 Jan. 2000; and EP 00870280.5, filed 22 Nov. 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to the diagnosis of tauopathies.The present invention provides a new method for the detection and/ordifferential diagnosis of tauopathies. The present invention alsoprovides a phospho-peptide that can be used for standardization in amethod of the invention.

BACKGROUND OF THE INVENTION

[0003] Several forms of dementia, the so-called tauopathies (Goedert etal., 1998), have been associated with the same pathophysiologicalmechanism, the involvement of the structural protein tau. Themicrotubule-associated protein tau is for example a major proteincomponent of paired helical filaments (PHF) and neurofibrillar tangles(NFT), associated with Alzheimer's disease (Brion et al., 1985;Delacourte and Defossez, 1986;

[0004] Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al.,1986; Kondo et al., 1988). Tau protein exists in different isoforms, ofwhich 4 to 6 are found in adult brain but only 1 isoform is detected infetal brain. The diversity of the isoforms is generated from a singlegene on human chromosome 17 by alternative mRNA splicing (Himmler, 1989;Goedert et al., 1989; Andreadis et al., 1992). The most striking featureof tau protein, as deduced from molecular cloning, is a stretch of 31 or32 amino acids, occurring in the carboxy-terminal part of the molecule,which can be repeated either 3 or 4 times. Additional diversity isgenerated through 29 or 58 amino acid-long insertions in theNH₂-terminal part of tau molecules (Goedert et al., 1989). In vivo taupromotes microtubule assembly and stability in the axonal compartment ofneurons by interactions involving its microtubule binding domain whichis localized in the repeat region of tau (255-381) (Lewis et al., 1988).In normal circumstances adult brain contains 2-3 mole phosphate per moleof tau (Selden and Pollard, 1983; Ksiezak-Reding et al., 1992).Phosphorylation of different sites in normal tau as studied in rat andhumans is dependent on the developmental state (Lee et al., 1991;Bramblett et al., 1993; Goedert et al., 1993). Tau variants of 60, 64and 68 kDa arising as a consequence of phosphorylation have beendetected in brain areas showing neurofibrillary tangles (Delacourte etal., 1990; Goedert et al., 1992; Flament et al., 1990, Greenberg andDavies, 1990). These brains contain 6-8 mole phosphate per mole tau(Ksiezak-Reding et al., 1992). In tau isolated from PHF (PHF-tau),phosphorylation occurs at several positions (Iqbal et al., 1989; Lee etal., 1991; Hasegawa et al., 1992; Hanger et al., 1998; Buee et al.,1999).

[0005] Alzheimer's disease (AD) is the most common type of primarydegenerative dementia associated with a tau pathology, having aprevalence of 42-75% (Brun, 1993; Gustafson, 1993; Ebly et al., 1994).Frontotemporal dementia (FTD) is a clinical condition in whichpathologically Pick's disease, Frontotemporal dementia with Parkinsonismlinked to chromosome 17, sporadic FTD and motor neuron disease arepresent. According to a small study by Mann et al. (2000), 16 of the 37cases with FTD could be classified as tauopathy based ontau-immunohistochemistry. Filamentous tau pathology i.e. neurofibrillarytangles (NFT), are consistently found in AD (Tomlinson and Corsellis,1984) but may also be found in FTD (Spillantini and Goedert, 1998).Pathological tau proteins are found both in AD and FTD (Vermersch etal., 1995; Delacourte et al., 1996), however studies on brain tissuehave suggested that the tau pathology differs between AD and FTD,possibly being related to the degree of phosphorylation (Delacourte etal., 1996). Other forms of dementia associated with a tau pathologyinclude Progressive supranuclear palsy (PSP), Corticobasal degeneration(CBD) and Subacute sclerosing panencephalitis. The role ofhyperphosphorylation in the pathology of these tauopathies is at presentnot well understood. In addition, various difficulties have beenencountered in the accurate determination of the degree ofphosphorylation of specific phospho-sites concentrated in the prolineregion. Because of these difficulties, an accurate method for thespecific detection of these tauopathies is still lacking.

SUMMARY OF THE INVENTION

[0006] The present invention relates to a method for the diagnosis of atauopathy in an individual, said method involving:

[0007] determining the ratio of phospho-tau (181)/total tau in saidindividual;

[0008] inferring that said individual is suffering a tauopathy bycomparing the obtained ratio of phospho-tau (181)/total tau in saidindividual with the ratio of phospho-tau (181)/total tau in controlindividuals, whereby an altered ratio of phospho-tau (181)/total taucompared to said ratio in control individuals being an indication oftauopathy.

[0009] The present invention also relates to a method for thedifferential diagnosis of a tauopathy versus a non-tauopathy in anindividual, said method involving:

[0010] determining the ratio of phospho-tau (181)/total tau in saidindividual; inferring that said individual is suffering a tauopathy bycomparing the obtained ratio of phospho-tau (181)/total tau in saidindividual with the ratio of phospho-tau (181)/total tau in individualssuffering a non-tauopathy or with the phospho-tau (181)/total tau ratioin control individuals, whereby an altered ratio of phospho-tau(181)/total tau compared to said ratio in individuals suffering anon-tauopathy or in control individuals being an indication oftauopathy.

[0011] It is an aim of the present invention to provide a method for thediagnosis of a tauopathy in an individual.

[0012] It is another aim of the present invention to provide a methodfor the diagnosis of Alzheimer's disease, Pick's disease, sporadicFrontotemporal dementia and/or Frontotemporal dementia with Parkinsonismlinked to chromosome 17 in an individual.

[0013] It is another aim of the present invention to provide a methodfor the differential diagnosis of a tauopathy versus a non-tauopathy.

[0014] It is another aim of the present invention to provide a methodfor the differential diagnosis of a tauopathy versus a non-tauopathyneurodegeneration.

[0015] It is another aim of the present invention to provide a methodfor the differential diagnosis of a tauopathy versus vascular dementia,Creutzfeldt Jacob Disease, stroke and/or neurotoxicity in patients withleukemia.

[0016] It is another aim of the present invention to provide a methodfor the differential diagnosis of Alzheimer's disease, Pick's disease,sporadic Frontotemporal dementia and/or Frontotemporal dementia withParkinsonism linked to chromosome 17 versus vascular dementia,Creutzfeldt Jacob Disease, stroke and/neurotoxicity in patients withleukemia.

[0017] It is another aim of the present invention to provide an in vitromethod as described above.

[0018] It is another aim of the present invention to provide aphospho-peptide for use in standardization.

[0019] It is another aim of the present invention to provide aphospho-peptide for use in standardization in a method to detectphospho-tau (181).

[0020] It is another aim of the present invention to provide aphospho-peptide for use in standardization in a method as describedabove.

[0021] It is another aim of the present invention to provide adiagnostic kit for use in a method as described above.

[0022] It is another aim of the present invention to provide a peptide,a method and/or a diagnostic kit for the testing or screening of drugs,for therapeutic monitoring and/or for the determination of theeffectiveness of a certain treatment for a tauopathy.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1. Fine mapping of tau antibodies, HT7, tau1, BT2, AT120 andAT270 on overlapping synthetic peptides. Only immunoreactive peptidesare shown. A. Mapping on peptides synthesized on pins. 48 nonapeptidesoverlapping 8 amino acids were used to cover the tau region from 155till 208. B. Peptide synthesis on paper. Sixteen overlapping peptides,12 amino acids long define the AT120 epitope in the region 206-232. C.Mapping of the AT270 antibody on biotinylated phosphopeptides (thephosphorylated threonine is indicated) covering the region 166 until196.

[0024]FIG. 2. Specificity of the AT270 antibody for phospho-Thr 181 asdefined by screening synthetic phosphopeptides. Biotinylatedphosphopeptides were captured on streptavidin-coated plates at aconcentration of 1 μg/ml. AT270 was detected via a peroxidase-coupledsecondary antibody. The sequences of the peptides are shown in Table 1.

[0025]FIG. 3. Relationship of phospho-tau levels, determined by theHT7-AT270 assay, and total tau levels (AT120-(HT7-BT2) assay). SeveralPHF-tau preparations were assayed at different dilutions. The figureshows the raw data as assayed in duplicate on two different plates fromthe dilution of the PHF-tau preparation which corresponds toapproximately 0.5 OD₄₅₀. Bars on the graph are standard deviations.PHF-A through D are derived from frontotemporal cortex, while PHF-E isprepared from a hippocampal region of an Alzheimer brain.

[0026]FIG. 4. Scatterplot of CSF-total tau in frontotemporal dementia,Parkinson's disease, Alzheimer's disease and subcorticalartheriosclerotic encephalopathy.

[0027]FIG. 5. Scatterplot of CSF-phospho-tau (181) in frontotemporaldementia, Parkinson's disease, Alzheimer's disease and subcorticalartheriosclerotic encephalopathy.

[0028]FIG. 6. Plot of correlation between CSF-total tau andCSF-phospho-tau (181), with all individuals in the study included.

[0029]FIG. 7. Scatterplot of phospho-tau (181)/total tau ratio in CSFfrom patients with frontotemporal dementia, Parkinson's disease,Alzheimer's disease and subcortical artheriosclerotic encephalopathy.

[0030]FIG. 8. CSF-total-tau (left) and CSF-phospho-tau (181) (right) atdifferent time points after acute stroke. Staples are means and bars SD.Number of samples at different time points: day 0-1: n=9, day 2-3: n=18,day 7-9: n=22, week 3: n=21, month 3-5: n=21. Significance compared withday 0-1: for CSF-tau: day 2-3: p=0.002, day 7-9: p<0.0001, week 3:p<0.0001, month 3-5: p=0.035; for CSF-phospho-tau: No significantdifferences at any time point compared with day 0

[0031]FIG. 9. Individual values for CSF-total-tau (left) andCSF-phospho-tau (181) (right) at different time points for the ninepatients of which CSF samples were taken at day one.

[0032]FIG. 10. Correlation between CSF-total-tau and CSF-phospho tau(181) in patients with Alzheimer's disease (n=54) and controls (n=17)(left), and in patients with acute stroke (n=22, number of samples=91)(right). TABLE 1 Sequence of the phosphorylated peptides used todetermine the specificity of AT270 for phospho-Thr 181. Name SequenceSEQ ID NO 153LysGlyAlaAspGlyLysThrLysIleAlaThr(p)ProArgGlyAlaAlaProProGlyGlnLys 8 175GlnAlaAsnAlaThrArgIleAlaProLysThr(p)ProProAlaProLysThrProProSerSer 9 181ArgIleProAlaLysThrProProAlaProLysThr(p)ProProSerSerGlyGluProProLysSer 10198 ProProLysSerGlyAspArgSerGlyTyrSer(p)SerGlySerProGlyThrProGlySerArg11 199ProLysSerGlyAspArgSerGlyTyrSerSer(p)GlySerProGlyThrProGlySerArgSer 12202 SerGlyAspArgSerGlyTyrSerSerGlySer(p)ProGlyThrProGlySerArgSerArgThr13 205ArgSerGlyTyrSerSerGlySerProGlyThr(p)ProGlySerArgSerArgThrProSerLeu 14208 TyrSerSerGlySerProGlyThrProGlySer(p)ArgSerArgThrProSerLeuProThrPro15 210SerGlySerProGlyThrProGlySerArgSer(p)ArgThrProSerLeuProThrProThrArg 16212 SerProGlyThrProGlySerArgSerArgThr(p)ProSerLeuProThrProThrArgGluPro17 214GlyThrProGlySerArgSerArgThrProSer(p)LeuProThrProThrArgGluProLysLys 18217 GlySerArgSerArgThrProSerLeuProThr(p)ProThrArgGluProLysLysValAlaVal19 231 ArgGluProLysLysValAlaValValArgThr(p)ProLysSerProSerSerAlaLysSer20 235 LysLysValAlaValValArgThrProLysSer(p)ProSerSerAlaLysSerArgLeuGln21 262 ValLysSerLysIleGlySer(p)ThrGluAsnLeuLys 22 396ThrAspHisGlyAlaGluIleValTyrLysSer(p)ProValValSerAspThrSerProArgHis 23400 AlaGluIleValTyrLysSerProValValSer(p)AspThrSerProArgHisLeuSerAsnVal24 403IleValTyrLysSerProValValSerAspThr(p)SerProArgHisLeuSerAsnValSerSer 25404 TyrLysSerProValValSerAspThrSer(p)ProArgHisLeuSerAsnValSerSerThr 26409 ValValSerAspThrSerProArgHisLeuSer(p)AsnValSerSerThrGlySerIleAspMet27 412AspThrSerProArgHisLeuSerAsnValSer(p)SerThrGlySerIleAspMetValAspSer 28422 SerSerThrGlySerIleAspMetValAspSer(p)ProGlnLeuAlaThrLeuAlaAspGluVal29

[0033] TABLE 2 Clinical characteristics of patients involved in thestudy described in example 2. Diag- Gender Duration Degree of Albuminnosis N (M:F) Age dementia (y) dementia^(a) ratio^(b) FTD 18  5:13 65.5± 8.4 4.1 ± 3.2 17.7 ± 6.4  9.7 ± 2.9 Prob 41 13:28 73.8 ± 5.9 3.2 ± 1.817.6 ± 5.2  5.4 ± 1.8 AD Poss 19 11:8  78.9 ± 5.7 3.0 ± 2.3 21.9 ± 3.7 7.9 ± 2.9 AD SAE 17 12:5  75.8 ± 4.4 2.6 ± 2.2 22.1 ± 7.2 11.9 ± 7.2 PD15 11:4  69.9 ± 7.5 — —  6.8 ± 2.4 Con- 17  4:13 71.8 ± 4.2 — —  5.3 ±1.8 trols

[0034] TABLE 3 CSF-levels of total tau, phospho-tau (181) andphospho-tau (181)/total tau ratio in dementia disorders, Parkinson'sdisease and normal aging. Cerebrospinal fluid levels (pM) Phospho tauDiagnosis Total tau Phospho-tau (181) (181)/total tau FTD  9.74 ± 2.88 8.59 ± 3.88*** 0.88 ± 0.34*** Probable AD 20.01 ± 7.58*** 23.12 ±10.10** 1.16 ± 0.24*** Possible AD 16.34 ± 4.30*** 18.01 ± 5.86 1.10 ±0.20*** SAE  3.70 ± 2.29***  6.39 ± 5.59*** 1.96 ± 1.08 PD  7.45 ± 1.5914.07 ± 3.11 1.92 ± 0.42 Controls  8.33 ± 2.83 15.92 ± 5.72 1.95 ± 0.60

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention relates to a method for the diagnosis of atauopathy in an individual, said method comprising the step of:

[0036] determining the ratio of phospho-tau (181)/total tau in saidindividual;

[0037] inference that said individual is suffering a tauopathy bycomparing the obtained ratio of phospho-tau (181)/total tau in saidindividual with the ratio of phospho-tau (181)/total tau in controlindividuals, an altered ratio of phospho-tau (181)/total tau compared tosaid ratio in control individuals being an indication.

[0038] The present invention also relates to a method for thedifferential diagnosis of a tauopathy versus a non-tauopathy in anindividual, said method comprising the steps of:

[0039] determining the ratio of phospho-tau (181)/total tau in saidindividual;

[0040] inference that said individual is suffering a tauopathy bycomparing the obtained ratio of phospho-tau (181)/total tau in saidindividual with the ratio of phospho-tau (181)/total tau in individualssuffering a non-tauopathy or with the phospho-tau (181)/total tau ratioin control individuals, an altered ratio of phospho-tau (181)/total taucompared to said ratio in individuals suffering a non-tauopathy or incontrol individuals being an indication.

[0041] The present invention is based on the finding that the ratio ofphospho-tau (181)/total tau in CSF from patients suffering AD and in CSFfrom patients suffering certain forms of FTD is significantly alteredcompared to the phospho-tau (181)/total tau ratio in CSF from controlindividuals. The present invention is further based on the finding thatthe ratio of phospho-tau (181)/total tau in CSF from patients sufferingAD is significantly altered compared to the phospho-tau (181)/total tauratio in CSF from patients suffering stroke. The indication that thephospho-tau (181)/total tau ratio in patients with a tauopathy isaltered, forms a basis for the development of a diagnostic test for thediagnosis of a tauopathy in an individual and/or for the differentialdiagnosis of individuals suffering a tauopathy versus individualssuffering a non-tauopathy.

[0042] ‘A tauopathy’ is any form of dementia that is associated with atau pathology.

[0043] Alzheimer's disease and certain forms of Frontotemporal dementia(Pick's disease, sporadic Frontotemporal dementia and Frontotemporaldementia with Parkinsonism linked to chromosome 17) are the most commonforms of tauopathy. In accordance, the present invention relates to anymethod as described above, wherein the tauopathy is Alzheimer's, Pick'sdisease, sporadic Frontotemporal dementia and Frontotemporal dementiawith Parkinsonism linked to chromosome 17. Other tauopathies include butare not limited to Progressive supranuclear palsy (PSP), Corticobasaldegeneration (CBD) and Subacute sclerosing panencephalitis.

[0044] In a specific embodiment, the present invention relates to amethod for the diagnosis of Azheimer's disease in an individual, saidmethod comprising the steps of:

[0045] determining the ratio of phospho-tau (181)/total tau in saidindividual;

[0046] inference that said individual is suffering Alzheimer's diseaseby comparing the obtained ratio of phospho-tau (181)/total tau in saidindividual with the ratio of phospho-tau (181)/total tau in controlindividuals, an altered ratio of phospho-tau (181)/total tau compared tosaid ratio in control individuals being an indication.

[0047] A ‘non-tauopathy’ is any status of the brain that is notassociated with a tau pathology. In an embodiment of the invention, saidnon-tauopathy is a non-tauopathy neurodegeneration. A non-tauopathyneurodegeneration is any form of neurological disorder that is notassociated with a tau pathology. Non-tauopathy neurodegenerationsinclude but are not limited to vascular dementia, Creutzfeldt JacobDisease, stroke and/or neurotoxicity in patients with leukemia.

[0048] Therefore, in a specific embodiment, the present inventionrelates to a method for the differential diagnosis in an individual ofAlzheimer's disease versus stoke, said method comprising the steps of:

[0049] determining the ratio of phospho-tau (181)/total tau in saidindividual;

[0050] inference that said individual is suffering Alzheimer's diseaseand not stroke by comparing the obtained ratio of phospho-tau(181)/total tau in said individual with the ratio of phospho-tau(181)/total tau in individuals suffering a stroke, an altered ratio ofphospho-tau (181)/total tau compared to said ratio in individualssuffering a stroke being an indication.

[0051] Phospho-tau (181) includes all tau molecules that carry aphosphate on the threonine at position 181. The numbering with respectto the amino acid sequence refers to the longest tau isoform hTau40(Goedert et al., 1989).

[0052] Total tau refers to all forms of tau and includes tau in anystate of phosphorylation.

[0053] The present invention thus relates to tau and phospho-tau (181)for use as neurological markers for the diagnosis of a tauopathy and/orfor the differential diagnosis of a tauopathy versus a non-tauopathy.

[0054] Based on the level of phospho-tau (181) and total tau in anindividual, the ratio of phospho-tau (181)/total tau in said individualcan then be determined.

[0055] The ratio of phospho-tau (181)/total tau can be detected in vitroas well as in vivo.

[0056] The method for the in vitro detection of the ratio of phospho-tau(181)/total tau in an individual comprises the steps of:

[0057] obtaining a sample from said individual;

[0058] determining the ratio of phospho-tau (181)/total tau in saidsample;

[0059] inference that said individual is suffering a tauopathy bycomparing the obtained ratio of phospho-tau (181)/total tau in saidindividual with the phospho-tau (181)/total tau ratio in a sample fromindividuals suffering a non-tauopathy or with the phospho-tau(181)/total tau ration in a sample from control individuals, an alteredratio of phospho-tau (181)/total tau compared to said ratio inindividuals suffering a non-tauopathy or in control individuals being anindication.

[0060] The term ‘sample’ refers to any source of biological material,for instance body fluids, brain extract, peripheral blood or any othersample comprising phospho-tau (181) protein. In an embodiment of theinvention, the ratio of phospho-tau (181)/total tau is determined invitro by analysis of the ratio of phospho-tau (181)/total tau in a bodyfluid sample of the patient. The term ‘body fluid’ refers to all fluidsthat are present in the human body including but not limited to blood,lymph, urine and cerebrospinal fluid (CSF) comprising phospo-tau (181)protein. In another embodiment of the present invention the ratio ofphospho-tau (181)/total tau is determined in a cerebrospinal fluid (CSF)sample taken from the patient. In accordance, the present inventionrelates to a method as described above, comprising the steps of:

[0061] obtaining a cerebrospinal fluid sample from the individual;

[0062] determining the ratio of phospho-tau (181)/total tau in saidcerebrospinal fluid sample;

[0063] inference that said individual is suffering a tauopathy bycomparing the obtained ratio of phospho-tau (181)/total tau in saidindividual with the ratio of phospho-tau (181)/total tau in the CSF fromindividuals suffering a non-tauopathy or with the phospho-tau(181)/total tau ratio in the CSF from control individuals, an alteredratio of phospho-tau (181)/total tau compared to said ratio in the CSFfrom individuals suffering a non-tauopathy or in the CSF from controlindividuals being an indication.

[0064] Total tau can be quantified by any method known, including butnot limited to the use of antibodies or else by a functional assay(Bramblett et al., 1992). Any monoclonal or polyclonal antibody thatspecifically recognizes total tau may be used for the quantification oftotal tau. Antibodies recognizing normally and abnormally phosphorylatedtau include Alz50 (Ghanbari et al., 1990), HT7 (Mercken et al., 1992)and AT120 (Vandermeeren et al., 1993). But also other antibodies knownin the art which recognize total tau can be used. A very fast anduser-friendly method for the quantification of total tau is the INNOTESThTau-Ag (Innogenetics, Gent, Belgium). Phospho-tau (181) can bequantified by any method known in the art, including but not limited tothe use of antibodies. In a preferred embodiment, phospho-tau (181) isquantified by an immunoassay comprising at least the following steps:

[0065] obtaining a sample from the patient;

[0066] bringing said sample into contact with a monoclonal antibodyspecifically recognizing phospho-tau (181), under conditions beingsuitable for producing an antigen-antibody complex;

[0067] detecting the immunological binding of said antibody to saidsample.

[0068] In an even more preferred embodiment, phospho-tau (181) can bequantified by a sandwich ELISA comprising the following steps:

[0069] obtaining a sample from the patient;

[0070] bringing said sample into contact with a monoclonal antibody(primary antibody or capturing antibody) recognizing phospho-tau (181),under conditions being suitable for producing an antigen-antibodycomplex;

[0071] bringing said sample into contact with a monoclonal antibody(secondary antibody or detector antibody) specifically recognizingphospho-tau (181), under conditions being suitable for producing anantigen-antibody complex;

[0072] bringing the antigen-antibody complex into contact with a markereither for specific tagging or coupling with said secondary antibody,with said marker being any possible marker known to the person skilledin the art;

[0073] possibly also, for standardization purposes, bringing theantibodies in contact with a purified phospho-tau protein orphospho-peptide reactive with both antibodies.

[0074] Advantageously, the secondary antibody itself carries a marker ora group for direct or indirect coupling with a marker.

[0075] The expression ‘recognizing’, ‘reacting with’, ‘immunologicalbinding’ or ‘producing an antigen-antibody complex’ as used in thepresent invention is to be interpreted that binding between the antigenand antibody occurs under all conditions that respect the immunologicalproperties of the antibody and the antigen.

[0076] The expression ‘specifically recognizing’ as used in the presentinvention is to be interpreted that said antibody is capable of formingan immunological complex with phospho-tau (181) but not with a taumolecule that lacks the phosphorylation at threonine 181.

[0077] Any monoclonal antibody that specifically recognizes phospho-tau(181) can be used in said method for the quantification of phospho-tau(181). A preferred monoclonal antibody for use in the quantification ofphospho-tau (181) is AT270 (International application published under WO95/17429). But also other antibodies known in the art that specificallyrecognize phospho-tau (181) can be used.

[0078] For standardization purposes, a tau protein or peptidephosphorylated at threonine 181 can be used. This can be obtained by anymethod such as extraction from brain or in vitro phosphorylation ofnormal tau. Since it is difficult to determine accurately the degree ofphosphorylation of specific phospho-sites concentrated in the prolineregion, in an embodiment of the invention, a synthetic phospho-peptideis used for standardization. Said synthetic phospho-peptide should beable to form an immunological complex with the antibodies used in theimmunoassay.

[0079] The present invention thus also relates to a phospho-peptidecomprising at least two epitopes that are recognized by a monoclonalantibody, said phospho-peptide being liable to form an immunologicalcomplex with said monoclonal antibodies in a sandwich ELISA. Previouswork has shown that, although a peptide contains an epitope for acertain monoclonal antibody, said monoclonal antibody does not alwaysrecognize said peptide (DeLeys et al., 1996). The present inventors wereable to define a phospho-peptide with two epitopes such, that indeedsaid phospho-peptide is able to form an immunological complex with themonoclonal antibodies recognizing said epitopes. In addition, thepresent inventors were able to define both epitopes such that thephospho-peptide is able to form an immunological complex with themonoclonal antibodies recognizing said epitopes in a sandwich ELISA.

[0080] The term ‘peptide’ refers to a polymer of amino acids (aa) anddoes not refer to a specific length of the product. In an embodiment ofthe invention, the length for the phospho-peptide is between 15 and 100amino acid. In a preferred embodiment of the invention, thephospho-peptide contains 20 to 50 amino acids. In another preferredembodiment of the invention, the phospho-peptide contains 30 to 40 aminoacids.

[0081] The peptide of the invention can be produced by any method knownin the art such as classical chemical synthesis as described byHoubenweyl (1974) and Atherton and Shepard (1989), by any commerciallyavailable method such as described in the examples section, or by meansof recombinant DNA techniques as described by Sambrook et al. (1989).

[0082] A phospho-peptide is a peptide that carries a phosphate on atleast one amino acid. The use of the phospho-peptide of the inventionallowed the present inventors to determine the relation ofphospho-peptide to specific phospho isoforms and to assess the degree ofphosphorylation of specific phospho-sites (see example 1, 1.5). The useof the phospho-peptide of the invention will allow the quantification ofparticular molecular forms of tau in a standardized way.

[0083] Phosphorylated peptides can be made by any method known. They canbe made post-assembly, by reaction for example with di-t-butyldiisopropyl diisopropylphosphoaramidite and oxidation with t-butylhydroperoxide of unprotected serine and threonine residues. They canalso be made by incorporation of phosphorylated amino acids duringpeptide synthesis. Recently, new phosphorylated serine derivatives(N-α-Fmoc-O-benzyl-L-phosphoSer) are commercially available(Calbiochem-Novabiochem AG, San Diego, Calif. 92121) to synthesizedirectly phosphopeptides without post-assembly phosphorylation.

[0084] In an embodiment, the present invention relates to aphospho-peptide liable to form an immunological complex with monoclonalantibody HT7 and monoclonal antibody AT270, comprising at least:

[0085] the minimal epitope of HT 7: ProProGlyGlnLys (SEQ ID NO 1); and

[0086] the minimal epitope of AT270: ProProAlaProLysThr(p)Pro (SEQ ID NO2).

[0087] In an even more preferred embodiment, the present inventionrelates to a phospho-peptide as described above, comprising thefollowing sequence: (SEQ ID NO 3)ProArgGlyAlaAlaProProGlyGlnLysGlyGlnAlaAsnAlaThrArgIleProAlaLysThrProProAlaProLysThr(p)ProProSer SerGlyGlu

[0088] wherein ‘(p)’ signifies that threonine is phosphorylated, orvariant sequences on the condition that they still bind to the HT7 andAT270 monoclonal antibodies. The term ‘variant sequences’ refers to anyvariant or fragment of the peptide represented in SEQ ID NO 3, bysubstitution or deletion of one or more amino acids, which stillrecognizes the HT7 and AT270 monoclonal antibodies. The term does notspecifically refer to, nor does it exclude, post-translationalmodifications of the peptide such as glycosylation, acetylation,phosphorylation, modifications with fatty acid and the like. Included inthe definition are, for example, peptides containing one or moreanalogues of an amino acid (including unnatural amino acids), peptideswith substituted linkages, mutated versions, peptides containingdisulfide bounds between cysteine residues, biotinylated peptides aswell as other modifications known in the art.

[0089] The present invention further relates to the use of saidphospho-peptide in a method for measuring the level of phospho-tau(181).

[0090] The present invention further relates to the use of saidphospho-peptide in a method for the diagnosis of a tauopathy and/or forthe differential diagnosis of a tauopathy versus a non-tauopathy.

[0091] The present invention further relates to the use of saidphospho-peptide in a method for the diagnosis of Alzheimer's disease.

[0092] The present invention further relates to the use of saidphospho-peptide in a method for the differential diagnosis ofAlzheimer's disease versus stroke.

[0093] The method for the in vitro detection of the ratio of phospho-tau(181)/total tau in an individual can also be used for testing orscreening of drugs, for therapeutic monitoring and/or to evaluate theeffect of a certain treatment on the tauopathy in said individual.

[0094] The method for the early in vivo detection of the ratio ofphospho-tau (181)/total tau in an individual comprises the steps ofdetermining the ratio of phospho-tau (181)/total tau in said individualand comparing it to the ratio of phospho-tau (181)/total tau in controlhealthy individuals. In an embodiment, phospho-tau (181) and total taucan be quantified by in vivo imaging. Phospho-tau (181) and total taucan be quantified in situ by non-invasive methods including but notlimited to brain imaging methods described by Arbit et al. (1995),Tamada et al. (1995), Wakabayashi et al. (1995), Huang et al. (1996),Sandrock et al. (1996), Mariani et al. (1997). These in vivo imagingmethods may allow the localization and quantification of phospho-tau(181) and total tau, for example, by use of labeled antibodiesrespectively specifically recognizing phospho-tau (181) or recognizingtotal tau.

[0095] Phospho-tau (181) and total tau can also be used as markers forin vivo imaging for testing or screening of drugs, for therapeuticmonitoring and/or to evaluate the effect of a certain treatment on thetauopathy in said individual.

[0096] The present invention further relates to a diagnostic kit for thediagnosis of a tauopathy in an individual and/or for the differentialdiagnosis of a tauopathy versus a non-tauopathy comprising at least anantibody specifically recognizing phospho-tau (181).

[0097] In another embodiment, the present invention relates to adiagnostic kit as described above comprising at least:

[0098] an antibody specifically recognizing phospho-tau (181);

[0099] an antibody recognizing tau.

[0100] In another embodiment, the present invention relates to adiagnostic kit as described above comprising at least a phospho-peptideaccording to the invention.

[0101] In another embodiment, the present invention relates to adiagnostic kit as described above comprising at least:

[0102] an antibody specifically recognizing phospho-tau (181);

[0103] a phospho-peptide according to the invention.

[0104] In another embodiment, the present invention relates to adiagnostic kit as described above comprising at least:

[0105] an antibody specifically recognizing phospho-tau (181);

[0106] an antibody recognizing tau;

[0107] a phospho-peptide according to the invention.

[0108] A preferred kit for the diagnosis of a tauopathy in an individualis based on an immunoassay and comprises:

[0109] a monoclonal antibody (primary antibody) which forms animmunological complex with an epitope of phospho-tau (181);

[0110] a monoclonal antibody (secondary antibody) which specificallyrecognizes phospho-tau (181);

[0111] a marker either for specific tagging or coupling with saidsecondary antibody;

[0112] appropriate buffer solutions for carrying out the immunologicalreaction between the primary antibody and the test sample, between thesecondary antibody and the test sample and/or between the boundsecondary antibody and the marker;

[0113] a phospho-peptide according to the invention.

[0114] The present invention further relates to the use of a diagnostickit as described above for the diagnosis of a tauopathy in an individualand/or for the differential diagnosis of a tauopathy versus anon-tauopathy.

[0115] The present invention further relates to the use of a diagnostickit as described above for the diagnosis of Alzheimer's disease, Pick'sdisease, sporadic Frontotemporal dementia and/or Frontotemporal dementiawith Parkinsonism linked to chromosome 17.

[0116] The present invention further relates to the use of a diagnostickit as described above for the differential diagnosis of Alzheimer'sdisease, Pick's disease, sporadic Frontotemporal dementia and/orFrontotemporal dementia with Parkinsonism linked to chromosome 17 versusvascular dementia, Creutzfeldt Jacob Disease, stroke and/orneurotoxicity in patients with leukemia.

[0117] The present invention also relates to the use of total tau andphospho-tau (181) as neurological markers for the manufacture of adiagnostic kit for the diagnosis of a tauopathy and/or for thedifferential diagnosis of a tauopathy versus a non-tauopathy.

[0118] The present invention also relates to the use of aphospho-peptide, a method and/or a diagnostic kit of the invention forthe testing or screening of drugs, for therapeutic monitoring and/or forthe determination of the effectiveness of a certain treatment for atauopathy.

[0119] Throughout this specification and the claims which follow, unlessthe context requires otherwise, the word “comprise”, and variations suchas “comprises” and “comprising”, will be understood to imply theinclusion of a stated integer or step or group of stated integers orsteps but not to the exclusion of any other integer or step or group ofintegers or steps.

[0120] The reference to any prior art in this specification is not, andshould not be taken as an acknowledgment or any form of suggestion thatthat prior art forms part of the common general knowledge in Australia.

[0121] The present invention will now be illustrated by reference to thefollowing examples that set forth particularly advantageous embodiments.However, it should be noted that these examples are illustrative and cannot be construed as to restrict the invention in any way.

EXAMPLES Example 1 Design of a Phospho-Peptide for use inStandardization

[0122] 1.1 Synthesis of Tau and Tau-Derived Peptides

[0123] Two PCR primers (a primer containing the starting methioninecodon—CATGGCTGAGCCCCGCCAGGAGTTCGAAGTGATGG (−1 to 34) (SEQ ID NO 4) andthe reverse primer around the stop codonCCTGATCACAAACCCTGCTTGGCCAGGGAGGC (SEQ ID NO 5)) were used to amplify thesmallest form from human tau into a P_(L)-based expression system(Innogenetics, Gent, Belgium). The sequence of the PCR product wasconfirmed by sequencing. Changes were only observed at the thirdbasepair in codons: at Pro 182 CAG instead of CAA, at Ala 227 GCGinstead of GCA, and at Asn 251 AAC instead of AAT. All numbering withrespect to the amino acid sequence refers to the longest tau isoform:hTau40 (Goedert et al., 1989). Deletion mutants were made based onconstructing frameshift mutants by filling in the SacII site (amino acidposition 154-155) and the PstI site (position 242-243).

[0124] Automated peptide synthesis was performed on a Millipore 9050synthesizer, usually as N-terminally biotinylated peptides. Large-scalesynthesis of peptideAcProArgGlyAlaAlaProProGlyGlnLysGlyGlnAlaAsnAlaThrArgIleProAlaLysThrProProAlaProLysThr(p)ProProSerSerGlyGlu-NH₂ (position 154-187) (SEQ ID NO 3)for sandwich ELISA was synthesized in house and by Neosystems(Strasbourg, France). Quality control includes RP-HPLC (reverse-phasehigh-pressure liquid chromatography) (>99% pure), mass-spectrometeranalysis (average MW 3454.8), and amino acid analysis (Net peptidecontent 84.3%).

[0125] For epitope mapping, peptides were synthesized manually onderivatized pins (Multiple Peptide Systems, San Diego, Calif. 92121) oron paper. For peptides synthesized on paper, the paper was derivatizedusing the symmetric anhydride of Fmoc-β-alanine(9-fluorenylmethoxycarbonyl-β-alanine) in the presence of dimethylaminopyridine. After removal of the Fmoc group, a second β-alanineresidue was added following activation with TBTU(═2-(1H-benzotriazole-1-yl)-1,1′,3,3′-tetramethyluroniumtetrafluoroborate). Peptides were subsequently synthesized manually asspots.

[0126] Phosphorylated peptides were made by post-assembly reaction withdi-t-butyl diisopropyl diisopropylphosphoaramidite and oxidation witht-butyl hydroperoxide of unprotected serine and threonine residues.Recently, new phosphorylated serine derivatives(N-α-Fmoc-O-benzyl-L-phosphoSer) are commercially available(Calbiochem-Novabiochem AG, San Diego, Calif. 92121) to synthesizedirectly phosphopeptides without post-assembly phosphorylation. Afterdetachment from the solid support, peptides and phosphopeptides werepurified by reverse-phase high-performance liquid chromatography(RP-HPLC). The quality of the peptides was verified by massspectrometry.

[0127] 1.2 Immunoassays

[0128] Details of the isolation and characterization of antibodies havebeen described for AT120 (Vandermeeren et al., 1993b), HT7 (Mercken etal., 1992), BT2 (Vandermeeren et al., 1993a) and AT270 (Goedert et al.,1994). To quantify peptide-antibody interactions by capturing assay,streptavidin (Roche Diagnostics, Brussels, Belgium) was coated at 5μg/ml overnight in 50 mM carbonate buffer, pH 9.5. After blocking,biotinylated peptides were added and detected with the tau antibodies. Asecond antibody coupled to horseradish peroxidase was used to quantifythe immunoreaction.

[0129] The research version of the INNOTEST phospho-tau (181P) wasdesigned as follows: HT7-coated immunoplates were incubated overnight at4° C. with 75 μl sample or standard, simultaneously with biotinylatedAT270. After washing, horseradish peroxidase-labeled streptavidin (RDI,Flanders, N.J., US) is added for 30 min. The reaction is stopped byaddition of 50 μl 0.9 N H₂SO₄.

[0130] Total tau was measured using the INNOTEST hTau-Ag, and acalculated molecular weight of 41065 for recombinant tau, which was usedas standard was employed to convert pg/ml to pM.

[0131] 1.3 Mapping of Tau Antibodies

[0132] In order to map the tau antibodies recognizing recombinant tau onthe complete tau molecule, deletion mutants were constructed based onthe PstI and SacII sites. The antibodies tested, i.e. HT7, AT120, BT2and tau1, all map to the proline-rich region (position 154-242 onhTau40, results not shown). To further delineate the epitopes, smalloverlapping peptides were synthesized. In a first step, 48 peptides, 9amino acids long and overlapping 8 amino acids were synthesized on pins.The sequence ranged from 155 until 208. AT120, HT7, BT2 and tau1 weretested. Four of the five antibodies could be mapped: the minimal epitopeof HT7 was ProProGlyGlnLys (position 159-163) (SEQ ID NO 1), while thereactivities of BT2 and tau-1 were indistinguishable: AspArgSerGlyTyrSer(position 193-198) (SEQ ID NO 6) (FIG. 1a). Since AT120 could not bemapped on these peptides, a new set of peptides was synthesized onpaper, covering the sequence from 206-232. A total of 16 peptides, 12amino acids-long, and overlapping with 11 amino acids, were needed tocover this region. The minimal epitope of AT120 was defined by thesequence ProProThrArgGluProLys (position 218-224) (SEQ ID NO 7) (FIG.1b).

[0133] 1.4 Specificity of AT270

[0134] The specificity of the phospho-dependent antibody, AT270, wasconfirmed on synthetic phospho-peptides covering 22 phosphorylationsites on tau. The sequences of these peptides are summarized in Table 1.Non-phosphorylated peptides corresponding to 12 of these sites wereanalyzed in parallel (FIG. 2). AT270 only reacted with phospho-peptidescontaining phospho-Thr 181 and/or phospho-Thr 175. When these peptideswere titrated out in a capturing assay, AT270 was 18-fold less reactiveon a molar basis with the peptide containing phospho-Thr 175 comparedwith that of phospho-Thr 181 (results not shown). Finally the minimalepitope was defined using biotinylated phosphorylated peptides 15 aminoacids-long covering the region 166-196. Immunoreactive peptides areshown in FIG. 1c, and the minimal epitope of AT270 wasProProAlaProLysThr(p)Pro (position 176-182) (SEQ ID NO 2).

[0135] 1.5 Design of Phospho-Peptide and Determination of the Degree ofPhosphorylation

[0136] Using this peptide information, a phospho-peptide was synthesizedcovering the position 154 to 187. This sequence covers the epitope ofHT7 (159-163) and AT270 (176-182) and an additional 5 amino acidsN-terminal to the HT7 epitope and C-terminal of the AT270 epitope. Theexact concentration of the phospho-peptide was determined using aminoacid analysis (Blennow et al., 1995). Based upon this concentration, thedynamic range of a peroxidase-based ELISA is between 5 and 300 pM usingprecision profiling. The intra-assay and interassay coefficients ofvariation were below 10%.

[0137] To determine how the degree of phosphorylation relates toabsolute levels of phospho-tau (181) and total tau, five differentPHF-tau preparations were simultaneously quantified in the respectiveassays. PHF-tau was prepared according to Goedert et al. (1992) using 1%N-lauroylsarcosinate to selectively precipitate PHF-tau from a brainextracts supernatants. Tissue from temporal cortex (PHF-A-D) orhippocampus (PHF-E) of AD patients was obtained from the Born-BungeBrain bank (Dr P. Cras, Antwerp, Belgium).

[0138] As shown in FIG. 3, the degree of phosphorylation of phospho-Thr181 was different between the PHF-tau preparations. Assuming that thePHF-tau preparation with the highest phospho-tau levels has a degree ofphosphorylation close to 100%, the ratio phospho-tau (181)/total tauoverestimates the degree of phosphorylation at least 3.3-fold.Nevertheless, taking into account that the ratio of phospho-tau(181)/total tau overestimates the degree of phosphorylation, thephosphorylation status of Thr 181 in tau from CSF-tau is 59%±18%(1.952/3.3) at most, which is in close agreement with thephosphorylation status of Thr 181 on brain-derived tau under normalconditions (20-30%; Watanabe et al., 1993; Matsuoet al., 1994).

Example 2 Use of the Phospho-Peptide for Standardization in an Assay toDetermine Phospho-Tau (181) in Patients with Alzheimer's Disease,Frontotemporal Dementia and/or Vascular Dementia

[0139] 2.1 Patients

[0140] Included in the study were 18 patients with FTD (age range 48-77years), 60 patients with AD (age range possible 68-88 years; probable58-90 years), 17 patients with subcortical artherioscleroticencephalopathy (SAE; a putative form of vascular dementia) (age range67-84 years), 15 patients with PD (age range 59-82 years), and 17controls (age range 68-80 years). Their characteristics are summarizedin table 2.

[0141] All patients included in the study had a clinical diagnosis ofFTD, AD, SAE, PD and were consecutively recruited from prospectivelongitudinal studies on patients with dementia or PD. Clinical diagnoseswere established and CSF sampling was performed. Then neurochemicalanalyses were performed at the Institute of Clinical Neuroscience,Sahlgrenska University Hospital, Mölndal, Sweden. Patients withunspecified dementia (e.g. mixed dementia), a history of severepsychiatric disease (e.g. schizophrenia), chronic alcoholism, distinctnon-degenerative neurological disease (e.g. normotensive hydrocephalus),a history of severe head injury, severe infections in the CNS, systemicdisease (e.g. malignant tumors) and secondary causes (e.g.hypothyreosis) for dementia according to the Diagnostic and statisticalmanual of mental disorders (Association AP, 1987) or biochemicalcriteria were excluded. Excluded were also patients with large cerebralinfarcts and/or multiple lacunas. All included patients underwent athorough clinical investigation, including medical history, physical,neurological and psychiatric examinations, screening laboratory tests ofblood (relevant laboratory tests to exclude other causes of ementia e.g.hypothyroidism), routine analysis of the CSF (e.g. cytology), ECG, chestX-ray, EEG, computerized tomography (CT) or magnetic resonance imaging(MRI) of the brain, investigation of the regional cerebral blood flow(CBF), using either single photon emission computerized tomography(SPECT) or ¹³³Xenon inhalation technique (Cortexplorer; Risberg andGustafson, 1983).

[0142] FTD was diagnosed according to the Lund/Manchester criteria (Brunet al., 1994) as previously described (Sjögren et al., 1997). None ofthe FTD patients had signs of infarcts, and only mild white-matterchanges were found in some FTD patients.

[0143] The diagnosis of ‘probable AD’ was made by exclusion, inaccordance with the NINCDS-ADRDA criteria (Mc Khann et al., 1984). TheAD patients were divided into one group with probable AD and one withpossible AD, as defined by the NINCDS-ADRDA criteria.

[0144] The diagnostic criteria for SAE were all of the following: a)mental deterioration (predominantly asteno-emotional disorder andfrontal cognitive dysfunction; b) gait disturbance (ataxia and/or motordysfunction); c) focal neurological signs; d) vascular risk factors suchas hypertension and diabetes, or presence of systemic vascular disease;e) at MRI or CT, bilateral multiple of diffusesubcortical-paraventricular deep white matter changes (>2 mm), lacunarinfarctions, an enlarged ventricular system and absence of more than onecortical infarction. The criteria were compatible with those suggestedby others (Bennett et al., 1990). Fifteen of the SAE patients had nocortical infarction; the remaining four had one cortical infarction.

[0145] The diagnosis of PD was made according to recommendations byLangstron and Koller (1991a, b). All the PD patients showed at least twoof the three features bradykinesia, rigidity and resting tremor, and allthe PD patients were responsive to L-dopa treatment. No patients with PDshowed any signs of dementia, and they all had a Mini-Mental StateExamination (MMSE) (Folstein et al., 1975) score of 27 or above.

[0146] All the clinical diagnoses were made by physicians withoutknowledge of the results of the biochemical analyses and vice versa.None of the patients were currently treated for dementia (e.g. withcholinesterase inhibitors).

[0147] In the demented patients, the degree of dementia was evaluatedusing the MMSE (Folstein et al., 1997).

[0148] The normal controls and the patients with PD were included in theanalysis of the sensitivity and specificity of the potential marker.

[0149] The control group consisted of individuals without history,symptoms or signs of psychiatric or neurological disease, malignantdisease or systemic disorders (e.g. rheumatoid arthritis, infectiousdisease). MMSE was used to evaluate their cognitive status, and thosewith scores below 28 were excluded.

[0150] The Ethics Committees of the Universities of Göteborg, Lund/Malmöand Linköping, Sweden, approved the study. All the patients (or theirnearest relations) and controls gave their informed consent toparticipating in the study, which was conducted in accordance with theprovisions of the Helsinki Declaration.

[0151] 2.2 CSF Analyses

[0152] On all patients and controls, lumbar puncture was performed atthe L3/L4 or L4/L5 interspace. The first 12 mL of CSF was collected inpolypropylene tubes and gently mixed to avoid gradient effects (Blennowet al., 1993). At the same time, a serum sample was taken. All CSFsamples with more than 500 erythrocytes per μl were excluded. The CSFand serum samples were centrifuged at 2000×g for 10 min. to eliminatecells and other insoluble material. Aliquots were then stored at −80° C.until biochemical analysis.

[0153] Quantitative determination of serum and CSF albumin was performedby nephelometry, using the Behring Nephelometer Analyzer (BehringwerkeAG, Marburg, Germany). The CSF/serum albumin ratio (Tibbling et al.,1977) was calculated as [CSF-albumin (mg/L)/serum-albumin (g/L)] and wasused as the measure of blood-brain barrier (BBB) function.

[0154] CSF-tau was determined using a sandwich ELISA (INNOTEST hTau-Ag,Innogenetics, Gent, Belgium), constructed to measure total tau (bothnormal tau and PHF-tau), as described previously in detail (Vandermeerenet al., 1993b; Blennow et al., 1995).

[0155] The level of phospho-tau (181) was determined by the INNOTESTphospho-tau (181P) as described above.

[0156] 2.3 Statistical Analysis

[0157] All variables were normally distributed and therefore parametricstatistics were used for group comparisons regarding the effectvariables (CSF-tau and CSF-phospho-tau). A fully factorial multipleANOVA was performed with CSF-tau and CSF-phospho-tau respectively asdependent variables, age, duration and severity of dementia ascovariates, and diagnostic category (probable and possible AD, FTD, PD,SAE, and normal aging) as factor. Factors that did not contribute to thevariance were excluded from the analysis and recalculation wasperformed. Post-hoc comparisons were performed using Turkey's post-hoctest for unequal n's.

[0158] The mean age was significantly lower in PD (p<0.001) and probableAD (p<0.05) compared to possible AD. Patients with probable AD weresignificantly more demented than patients with possible AD (p<0.05). Nodifferences were found between any of the patient or control groupsregarding the CSF/serum albumin ratio (for the dementia groups only),and gender (table 2).

[0159] 2.4 CSF-Total Tau and CSF-Phospho-Tau (181) in Dementia,Parkinson's Disease and Normal Aging

[0160] CSF-tau was significantly increased in probable AD and possibleAD compared to FTD (p<0.001), PD (p<0.001), SAE (p<0.001), and controls(p<0.001), and in FTD compared to SAE (p<0.01) (table 3; FIG. 4).

[0161] CSF-phospho-tau (181) was significantly increased in probable ADcompared to FTD (p<0.001), PD (p<0.001), SAE (p<0.001), and controls(p<0.0079), and in possible AD compared to FTD (p<0.001), and SAE(p<0.001), but not compared to controls. Furthermore, theCSF-phospho-tau (181) was also significantly decreased in FTD (p<0.0001)and in SAE (p<0.0001) as compared to controls (table 3; FIG. 5).

[0162] CSF-tau and CSF-phospho-tau (181) was positively correlated inall diagnostic groups (probable and possible AD: r=0.86, p<0.001; FTD:r=0.66, p<0.01; SAE: r=0.58, p<0.05; PD: r=0.62, p<0.05; controls:r=0.65, p<0.01; FIG. 6).

[0163] A CSF-phospho-tau (181)/CSF-total tau ratio was calculated andfound to be significantly decreased in probable AD (p<0.001; 1.16±0.24),possible AD (p<0.001; 1.10±0.20), and FTD (p<0.001; 0.88±0.34) comparedto controls (p<0.001 for all three groups; 1.95±0.60), PD (p<0.001 forall three groups; 1.92±0.42), and SAE (p<0.001 for all three groups;1.96±1.08) (table 3; FIG. 7).

Example 3 Determination of the CSF-Phospho Tau (181) and CSF-Total TauLevel in Patients with Alzheimer's Disease Versus Patients with AcuteIschemic Stroke

[0164] 3.1 Patients

[0165] CSF-total tau and CSF-phospho-tau (181) were examined inlongitudinal CSF samples from 22 patients, 16 men and 6 women, meanage±SD, 65.7±9.2 years, with an acute ischemic stroke. All patients wereevaluated in a standardized way, as described previously (Tarkowski etal., 1999). When possible, CSF samples were collected on five occasions;at income day 0-1 (n=9), day 2-3 (n=18), day 7-9 (n=22), three weeks(n=21) and 3-5 months (n=21).

[0166] Also 54 patients with probable AD, 25 men and 29 women, meanage+SD, 73.3±7.4 years, and 17 healthy controls, 4 men and 13 women,mean age+SD, 68.6±7.5 years were studied. The mean age was significantly(p<0.05) higher in the AD than in the control group. The diagnosis of“probable AD” was made by exclusion, according to the NINCDS-ADRDAcriteria (McKhann et al., 1984). The clinical evaluation and diagnosticprocedure have been described in detail elsewhere (Andreasen et al.,1998, 1999). The degree of dementia was evaluated using the MMSE(Folstein, 1975), and was 23.8+4.4 in the AD group. The control groupconsisted of individuals without history, symptoms or signs ofpsychiatric or neurological disease, malignant disease or systemicdisorders. Individuals with MMSE scores below 28 were not included. Allclinical diagnoses were made without knowledge of the results of thebiochemical analyses and vice versa.

[0167] The stroke patients were examined by computerized tomography (CT)and magnetic resonance imaging (MRI) about 1 month after stroke, asdescribed in detail previously (Tarkowski et al, 1999). The size (incm²) was determined by CT and the volume (in mL) by MRT.

[0168] The Kruskal-Wallis test was used for comparisons between three ormore groups, and if significant, the Mann-Whitney U-test for comparisonsbetween two groups. The Spearman correlation coefficient was used forcorrelations.

[0169] The Ethics Committees of the Universities of Göteborg and Umeåapproved the study. All patients (or their nearest relatives) andcontrols gave their informed consent to participate in the study, whichwas conducted according to the provisions of the Helsinki Declaration.

[0170] 3.2 CSF Analyses

[0171] A lumbar puncture was performed in the L3/L4 or L4/L5 interspace.The first 12 mL of CSF was collected in polypropylene tubes and gentlymixed to avoid possible gradient effects (Blennow, 1993). CSF sampleswith more than 500 erythrocytes per μl were excluded. The CSF sampleswere centrifuged at 2000×g for 10 min. to eliminate cells and otherinsoluble material, and aliquots were then stored at −80° C. pendingbiochemical analyses.

[0172] The level of CSF-tau was determined using a sandwich ELISA(Innotest hTAU-Ag, Innogenetics, Gent, Belgium), constructed to measuretotal tau (both normal tau and PHF-tau), as described in detailpreviously (Vandermeeren et al., 1993b; Blennow et al., 1995). The levelof CSF-phospho-tau (181) was determined by the INNOTEST phospho-tau(181P) as described above.

[0173] 3.3 CSF-Phospho Tau (181) and CSF-Total Tau in Patients withAlzheimer's Disease and in Patients with Stroke

[0174] In the stroke patients, CSF-total-tau showed a marked increase atday 2-3 (24.4+7.4 pM; p=0.002) after the acute stroke as compared withday 0-1 (mean+SEM 5.4+1.2 pM), and stayed elevated at day 7-9 (24.1+4.3pM; p<0.0001) and after three weeks (25.0+4.1 pM; p<0.0001) and thenreturned to normal levels after 3-5 months (8.5+1.2 pM; p=0.35) (FIG.8). The individual values for CSF-total tau for the nine patients withCSF samples taken at baseline are given in FIG. 9.

[0175] In contrast, there was no significant change in CSF-phospho-tau(181) between baseline (mean+SEM 9.1+3.1 pM) and day 2-3 (10.9+1.0 pM),day 7-9 (13.0+2.1 pM), three weeks (11.5+2.0 pM) or 3-5 months (10.6+2.0pM) (FIG. 8). The individual values for CSF-phospho-tau (181) for thenine patients with CSF samples taken at baseline are given in FIG. 9.

[0176] CSF-total tau was significantly increased in probable AD (mean+SD14.4+5.5 pM) compared to controls (8.3+2.8 pM) (p<0.0001). AlsoCSF-phospho-tau (181) was significantly increased in probable AD(20.5+6.5 pM) compared to controls (15.9+5.7 pM) (p<0.05).

[0177] CSF-total tau and CSF-phospho-tau (181) were positivelycorrelated in the AD (r=0.93; p<0.0001) and in the control (r=0.72,p<0.01) groups (FIG. 10). In the stroke group, the correlation washigher at day 0-1 (r=0.63, p<0.001) and after 3 months (r=0.80), than atday 2-3 (r=0.54), day 7-9 (r=0.58) and especially after three weeks(r=0.15).

[0178] From FIG. 10 it is clear that there is a difference between theratio CSF-phospho tau (181)/CSF-total tau for Alzheimer's diseasepatients compared to stroke patients. Regression analysis for valuesobtained from Alzheimer's disease patients (y=0.82x+4.39, r=0.90,p<0.0001) and from Stroke patients (y=0.17x+8.09, r=0.415, p<0.0001)revealed a significant difference (p<0.001).

[0179] There was a positive correlation between the size of the infarctas measured by CT and the maximum level of CSF-total tau (r=0.72;p<0.01), while the correlation to CSF-phospho-tau (181) was notsignificant (r=0.349). There was also a positive correlation between thevolume of the infarct as measured by MRI and the maximum value ofCSF-total tau (r=0.66; p<0.05), while the correlation to CSF-phospho-tau(181) was not significant (r=0.59).

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1 54 1 5 PRT Homo sapiens 1 Pro Pro Gly Gln Lys 1 5 2 7 PRT Homo sapiensMOD_RES (6)..(6) PHOSPHORYLATION 2 Pro Pro Ala Pro Lys Thr Pro 1 5 3 34PRT Homo sapiens MOD_RES (28)..(28) PHOSPHORYLATION 3 Pro Arg Gly AlaAla Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr 1 5 10 15 Arg Ile ProAla Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser 20 25 30 Gly Glu 435 DNA Homo sapiens 4 catggctgag ccccgccagg agttcgaagt gatgg 35 5 32 DNAHomo sapiens 5 cctgatcaca aaccctgctt ggccagggag gc 32 6 6 PRT Homosapiens 6 Asp Arg Ser Gly Tyr Ser 1 5 7 7 PRT Homo sapiens 7 Pro Pro ThrArg Glu Pro Lys 1 5 8 21 PRT Homo sapiens MOD_RES (11)..(11)PHOSPHORYLATION 8 Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro ArgGly Ala Ala 1 5 10 15 Pro Pro Gly Gln Lys 20 9 21 PRT Homo sapiensMOD_RES (11)..(11) PHOSPHORYLATION 9 Gln Ala Asn Ala Thr Arg Ile Ala ProLys Thr Pro Pro Ala Pro Lys 1 5 10 15 Thr Pro Pro Ser Ser 20 10 22 PRTHomo sapiens MOD_RES (12)..(12) PHOSPHORYLATION 10 Arg Ile Pro Ala LysThr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser 1 5 10 15 Gly Glu Pro ProLys Ser 20 11 21 PRT Homo sapiens MOD_RES (11)..(11) PHOSPHORYLATION 11Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Gly Ser Pro Gly 1 5 1015 Thr Pro Gly Ser Arg 20 12 21 PRT Homo sapiens MOD_RES (11)..(11)PHOSPHORYLATION 12 Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Gly SerPro Gly Thr 1 5 10 15 Pro Gly Ser Arg Ser 20 13 21 PRT Homo sapiensMOD_RES (11)..(11) PHOSPHORYLATION 13 Ser Gly Asp Arg Ser Gly Tyr SerSer Gly Ser Pro Gly Thr Pro Gly 1 5 10 15 Ser Arg Ser Arg Thr 20 14 21PRT Homo sapiens MOD_RES (11)..(11) PHOSPHORYLATION 14 Arg Ser Gly TyrSer Ser Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser 1 5 10 15 Arg Thr ProSer Leu 20 15 21 PRT Homo sapiens MOD_RES (11)..(11) PHOSPHORYLATION 15Tyr Ser Ser Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro 1 5 1015 Ser Leu Pro Thr Pro 20 16 21 PRT Homo sapiens MOD_RES (11)..(11)PHOSPHORYLATION 16 Ser Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg ThrPro Ser Leu 1 5 10 15 Pro Thr Pro Thr Arg 20 17 21 PRT Homo sapiensMOD_RES (11)..(11) PHOSPHORYLATION 17 Ser Pro Gly Thr Pro Gly Ser ArgSer Arg Thr Pro Ser Leu Pro Thr 1 5 10 15 Pro Thr Arg Glu Pro 20 18 21PRT Homo sapiens MOD_RES (11)..(11) PHOSPHORYLATION 18 Gly Thr Pro GlySer Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Thr 1 5 10 15 Arg Glu ProLys Lys 20 19 21 PRT Homo sapiens MOD_RES (11)..(11) PHOSPHORYLATION 19Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Thr Arg Glu Pro 1 5 1015 Lys Lys Val Ala Val 20 20 20 PRT Homo sapiens MOD_RES (11)..(11)PHOSPHORYLATION 20 Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro LysSer Pro Ser 1 5 10 15 Ser Ala Lys Ser 20 21 20 PRT Homo sapiens MOD_RES(11)..(11) PHOSPHORYLATION 21 Lys Lys Val Ala Val Val Arg Thr Pro LysSer Pro Ser Ser Ala Lys 1 5 10 15 Ser Arg Leu Gln 20 22 12 PRT Homosapiens MOD_RES (7)..(7) PHOSPHORYLATION 22 Val Lys Ser Lys Ile Gly SerThr Glu Asn Leu Lys 1 5 10 23 21 PRT Homo sapiens MOD_RES (11)..(11)PHOSPHORYLATION 23 Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro ValVal Ser Asp 1 5 10 15 Thr Ser Pro Arg His 20 24 21 PRT Homo sapiensMOD_RES (11)..(11) PHOSPHORYLATION 24 Ala Glu Ile Val Tyr Lys Ser ProVal Val Ser Asp Thr Ser Pro Arg 1 5 10 15 His Leu Ser Asn Val 20 25 21PRT Homo sapiens MOD_RES (11)..(11) PHOSPHORYLATION 25 Ile Val Tyr LysSer Pro Val Val Ser Asp Thr Ser Pro Arg His Leu 1 5 10 15 Ser Asn ValSer Ser 20 26 20 PRT Homo sapiens MOD_RES (10)..(10) PHOSPHORYLATION 26Tyr Lys Ser Pro Val Val Ser Asp Thr Ser Pro Arg His Leu Ser Asn 1 5 1015 Val Ser Ser Thr 20 27 21 PRT Homo sapiens MOD_RES (11)..(11)PHOSPHORYLATION 27 Val Val Ser Asp Thr Ser Pro Arg His Leu Ser Asn ValSer Ser Thr 1 5 10 15 Gly Ser Ile Asp Met 20 28 21 PRT Homo sapiensMOD_RES (11)..(11) PHOSPHORYLATION 28 Asp Thr Ser Pro Arg His Leu SerAsn Val Ser Ser Thr Gly Ser Ile 1 5 10 15 Asp Met Val Asp Ser 20 29 21PRT Homo sapiens MOD_RES (11)..(11) PHOSPHORYLATION 29 Ser Ser Thr GlySer Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr 1 5 10 15 Leu Ala AspGlu Val 20 30 78 PRT Homo sapiens 30 Arg Gly Ala Ala Pro Pro Gly Gln LysGly Gln Ala Asn Ala Thr Arg 1 5 10 15 Ile Pro Ala Lys Thr Pro Pro AlaPro Lys Thr Pro Pro Ser Ser Gly 20 25 30 Glu Pro Pro Lys Ser Gly Asp ArgSer Gly Tyr Ser Ser Pro Gly Ser 35 40 45 Pro Gly Thr Pro Gly Ser Arg SerArg Thr Pro Ser Leu Pro Thr Pro 50 55 60 Pro Thr Arg Glu Pro Lys Lys ValAla Val Val Arg Thr Pro 65 70 75 31 9 PRT Homo sapiens 31 Arg Gly AlaAla Pro Pro Gly Gln Lys 1 5 32 9 PRT Homo sapiens 32 Gly Ala Ala Pro ProGly Gln Lys Gly 1 5 33 9 PRT Homo sapiens 33 Ala Ala Pro Pro Gly Gln LysGly Gln 1 5 34 9 PRT Homo sapiens 34 Ala Pro Pro Gly Gln Lys Gly Gln Ala1 5 35 9 PRT Homo sapiens 35 Pro Pro Gly Gln Lys Gly Gln Ala Asn 1 5 369 PRT Homo sapiens 36 Lys Ser Gly Asp Arg Ser Gly Tyr Ser 1 5 37 9 PRTHomo sapiens 37 Ser Gly Asp Arg Ser Gly Tyr Ser Ser 1 5 38 9 PRT Homosapiens 38 Gly Asp Arg Ser Gly Tyr Ser Ser Pro 1 5 39 9 PRT Homo sapiens39 Asp Arg Ser Gly Tyr Ser Ser Pro Gly 1 5 40 12 PRT Homo sapiens 40 ProSer Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 1 5 10 41 12 PRT Homosapiens 41 Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys 1 5 10 42 12PRT Homo sapiens 42 Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val 1 510 43 12 PRT Homo sapiens 43 Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys ValAla 1 5 10 44 12 PRT Homo sapiens 44 Thr Pro Pro Thr Arg Glu Pro Lys LysVal Ala Val 1 5 10 45 12 PRT Homo sapiens 45 Pro Pro Thr Arg Glu Pro LysLys Val Ala Val Val 1 5 10 46 15 PRT Homo sapiens MOD_RES (14)..(14)PHOSPHORYLATION 46 Ala Thr Arg Ile Pro Ala Lys Thr Pro Pro Ala Pro LysThr Pro 1 5 10 15 47 15 PRT Homo sapiens MOD_RES (13)..(13)PHOSPHORYLATION 47 Thr Arg Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys ThrPro Pro 1 5 10 15 48 15 PRT Homo sapiens MOD_RES (12)..(12)PHOSPHORYLATION 48 Arg Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr ProPro Ser 1 5 10 15 49 15 PRT Homo sapiens MOD_RES (11)..(11)PHOSPHORYLATION 49 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro ProSer Ser 1 5 10 15 50 15 PRT Homo sapiens MOD_RES (10)..(10)PHOSPHORYLATION 50 Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro SerSer Gly 1 5 10 15 51 15 PRT Homo sapiens MOD_RES (9)..(9)PHOSPHORYLATION 51 Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser SerGly Glu 1 5 10 15 52 15 PRT Homo sapiens MOD_RES (8)..(8)PHOSPHORYLATION 52 Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser GlyGlu Pro 1 5 10 15 53 15 PRT Homo sapiens MOD_RES (7)..(7)PHOSPHORYLATION 53 Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly GluPro Pro 1 5 10 15 54 15 PRT Homo sapiens MOD_RES (6)..(6)PHOSPHORYLATION 54 Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu ProPro Lys 1 5 10 15

1. A method for measuring the level of phospho-tau (181) in a samplecomprising: a) detecting phospho-tau in a sample; and b) using aphospho-peptide as a standard to measure the level of phospho-tau (181);wherein the phospho-peptide is between 15 and 100 amino acids in length,wherein the phospho-peptide is liable to form an immunological complexwith monoclonal antibody HT7 and monoclonal antibody AT270, and whereinthe phospho-peptide comprises: the minimal epitope of HT 7:ProProGlyGlnLys (SEQ ID NO 1); and the minimal epitope of AT270:ProProAlaProLysThr(p)Pro (SEQ ID NO 2).
 2. The method of claim 1,wherein said phospho-peptide comprises the following sequence: (SEQ IDNO 3) ProArgGlyAlaAlaProProGlyGlnLysGlyGlnAlaAsnAlaThrArgIleProAlaLysThrProProAlaProLysThr(p)ProProSer SerGlyGlu

or variant sequences thereof on the condition that they still bind tothe HT7 and the AT270 monoclonal antibodies.
 3. The method of claim 2wherein the phospho-peptide comprises the sequence of SEQ ID NO:3. 4.The method of claim 2 wherein the phospho-peptide consists of thesequence of SEQ ID NO:3.
 5. The method of claim 2 wherein thephospho-peptide is from 20 to 50 amino acid residues in length.
 6. Themethod of claim 2 wherein the phospho-peptide is from 30 to 40 aminoacid residues in length.
 7. The method of claim 2 wherein the sample isa biological sample selected from the group consisting of: body fluidand brain extract.
 8. The method of claim 2 wherein the body fluid isselected from the group consisting of blood, lymph, urine, andcerebrospinal fluid.
 9. A diagnostic kit comprising a phospho-peptidewherein the phospho-peptide is between 15 and 100 amino acids in length,wherein the phospho-peptide is liable to form an immunological complexwith monoclonal antibody HT7 and monoclonal antibody AT270, and whereinthe phospho-peptide comprises: (a) the minimal epitope of HT 7:ProProGlyGlnLys (SEQ ID NO 1); and (b) the minimal epitope of AT270:ProProAlaProLysThr(p)Pro (SEQ ID NO 2).
 10. The diagnostic kit of claim9 wherein the phospho-peptide comprises the sequence: (SEQ ID NO 3)ProArgGlyAlaAlaProProGlyGlnLysGlyGlnAlaAsnAlaThrArgIleProAlaLysThrProProAlaProLysThr(p)ProProSer SerGlyGlu

or variant sequences thereof on the condition that they still bind tothe HT7 and the AT270 monoclonal antibodies.
 11. The diagnostic kit ofclaim 9 wherein the phospho-peptide comprises the sequence of SEQ IDNO:3.
 12. The diagnostic kit of claim 9 wherein the phospho-peptideconsists of the sequence of SEQ ID NO:3.
 13. The diagnostic kit of claim9 wherein the phospho-peptide is from 20 to 50 amino acid residues inlength.
 14. The diagnostic kit of claim 9 wherein the phospho-peptide isfrom 30 to 40 amino acid residues in length.
 15. A phospho-peptidecomprising: (a) the minimal epitope of HT 7: ProProGlyGlnLys (SEQ ID NO1); and (b) the minimal epitope of AT270: ProProAlaProLysThr(p)Pro (SEQID NO 2); wherein the phospho-peptide is between 15 and 100 amino acidsin length and wherein the phospho-peptide is liable to form animmunological complex with monoclonal antibody HT7 and monoclonalantibody AT270.
 16. The phospho-peptide of claim 15 that comprises thesequence (SEQ ID NO 3) ProArgGlyAlaAlaProProGlyGlnLysGlyGlnAlaAsnAlaThrArgIleProAlaLysThrProProAlaProLysThr(p)ProProSer SerGlyGlu

or variant sequences thereof on the condition that they still bind tothe HT7 and the AT270 monoclonal antibodies.
 17. The phospho-peptide ofclaim 15 comprising the sequence of SEQ ID NO:3.
 18. The phospho-peptideof claim 15 consisting of the sequence of SEQ ID NO:3.
 19. Thephospho-peptide of claim 15 that is from 20 to 50 amino acid residues inlength.
 20. The phospho-peptide of claim 15 that is from 30 to 40 aminoacid residues in length.